IS 1

The Function and Metabolism of Certain Insect Muscles inRelation to their Structure

By GEORGE A. EDWARDS AND HELMUT RUSKA

(From the Departamento de Fisiologia Geral e Animal, Universidade de Sao Paulo; Division ofLaboratories and Research, New York State Department of Health, Albany, New York; and

SecfSo de Virus, Institute Butantan, Sao Paulo, Brazil)

With 4 plates (figs. 2, 3, 6, and 7)

SUMMARY

Electron microscopic observations on ultrathin sections of the red thoracic flight-muscles and white leg muscles of Hydrophilus and Dytiscus are reported.

In red muscle-fibres with high values in frequency of contraction, oxygen consump-tion, and dehydrogenase activity, the single fibrils are completely surrounded by hugemitochondria. Tracheoles penetrate the sarcolemma and supply the mitochondria withoxygen by intracellular branches. In the less active white muscle fibres, mitochondriaare found irregularly scattered between the fibrils or along the I band. The intra-cellular tracheolization is sparse but an endoplasmic reticulum is widely spread betweenthe synfibrillar contractile material. The same muscles of the two insects differ con-siderably in detail.

INTRODUCTION

THE highly specialized functions of insect muscles make these the bestmodels for the study of the relationships between function, metabolism,

and structure. This had been noted by the earlier microscopists such asKnoll (1889) and Holmgren (1909, 1913), but in recent years has not receivedsufficient attention. Marked metabolic differences are to be found among thevarious muscles of various insects, these differences being closely related tothe function of the muscles and the phylogenetic position of the insect involved(see Perez Gonzalez and Edwards, 1954, for summary of the literature).Physiological studies of insect flight (Roeder, 1951) have also brought tolight considerable differences in nerve-muscle relationships between the higherand lower insects. Structural differences in the tracheation of muscle fibres,differences in colour of fibres, and variations of ultrastructure of isolated fibrilsalso occur from muscle to muscle in the insect (Edwards and others, 1954a,19546). In mammalian muscle (Ruska, 1954) and fowl-breast muscle (Bennettand Porter, 1953), electron microscope studies have suggested that the endo-plasmic reticular and mitochondrial systems may be closely related to theprocess of contraction within the fibril and further related to specializationin muscle function.

With these facts in mind we have begun to study ultrathin sections ofcertain insect muscles, with the electron microscope, to determine therelationship of metabolism and specialization in function to principles ofstructure. Particularly we have been interested in the relationship of thetracheoles to the mitochondria, and the roles played by the mitochondria[Quarterly Journal of Microscopical Science, Vol. 96, part 2, pp. 151-159, 1955.]

152 Edwards and Ruska—Function and Structure of Insect Muscles

and reticular systems in widely differing types of muscular functions, suchas flying and walking.

MATERIAL AND METHODS

The muscles used were the dorso-longitudinal, indirect, flight (red), andthe coxal levator (white) muscles of adult Hydrophilus ater and Dytiscus spp.The muscles were observed in both the contracted and stretched states. They

FIG. I . Red fibre of Hydrophilus. Many tracheoles outside and inside the thin sarcolemma,intermingled with the mitochondria between the contracted myofibrils and located close to

one nucleus.

were fixed in i per cent, buffered osmium tetroxide solution, according tothe method of Palade (1952), and embedded in methacrylate by the con-ventional method for electron microscopy. The sections were cut with amicrotome built in the Rockefeller Institute for Medical Research. Themicroscope used was the Siemens UM-ioob, of the Instituto Butantan inSao Paulo. From several micrographs, drawings were composed to show theoverall construction and to reduce the number of illustrations (see figs, i, 4,5, and 8). The originals were exhibited at the International Conference ofthe Joint Commission on Electron Microscopy, held at London University,16-21 July 1954.

FIG. 2 (plate). Red fibre of Hydrophilus. Electron micrograph. Four contracted fibrilsegments; m, mitochondria; t, tracheoles.

G. A. EDWARDS and H. RUSKA

G. A. EDWARDS and H. RUSKA

Edwards and Ruska—Function and Structure of Insect Muscles 153

RESULTS

Oxygen supply

The tracheoles have previously been thought to terminate on the surfaceof the muscle fibre. The red flight-muscle fibres have a large external trachealsupply; the white leg-muscle fibres have few tracheal branches. In ourpreparations we have seen that the tracheoles actually penetrate all musclefibres, but the number and distribution of penetrating tracheoles vary withthe type of muscle.

FIG. 4. Red fibre of Dytiscus. Few tracheoles outside and inside the thin sarcolemma; theyare attached to the closely packed mitochondria between the stretched myofibrils.

In the sections of Hydrophilus red fibres (figs. 1 and 2) it can be seen thatas the trachea approaches the fibre surface it gives off numerous fine branches(tracheoles) which penetrate the sarcolemma at various points. Just beneaththe sarcolemma may be seen many branches in cross, tangential, and longi-tudinal section. Between the fibrils, and closely associated with the mito-chondria, are found cross-sections of very fine, intracellular tracheolesthroughout the entire fibre. It is interesting to note that well in the interior

FIG. 3 (plate). Red fibre of Dytiscus. Electron micrograph. One stretched fibril segment;m, mitochondria; t, tracheoles.

154 Edwards and Ruska—Function and Structure of Insect Muscles

of the fibre the tracheoles.appear to be uniform in diameter; they are oftenin clusters, and always near or actually in contact with the mitochondria.The space between the fibrils in this muscle is considerable and appears tobe filled principally with the fine tracheoles and large mitochondria.

In the sections of the red flight-muscle of Dytiscus (figs. 3 and 4) fewertracheoles are observed. The general tracheal picture is similar to that of

FIG. 5. White fibre of Hydrophilus, Few tracheoles in cross-section beneath the sarco-lemma and between the mitochondria. The endoplasmic reticulum between the myofibrils

spreads along the Z bands, which are attached to the sarcolemma.

Hydrophilus, in the branching of the trachea at the surface and the penetrationby the finer tracheoles into the fibre. Differences appear in that there arefewer tracheoles within the fibre and they generally run longitudinally betweenthe fibrils, together with the mitochondria. Interestingly enough the tracheolediameter in this muscle is greater in relation to the fibril diameter than inthe Hydrophilus muscle. The mitochondria of this muscle are much moreclosely packed and this may well influence the tracheole distribution. Thus,it appears that in the red flight-muscle fibres of both insects the tracheoles

FIG. 6 (plate). White fibre of Hydrophilus. Electron micrograph. Fibrils; m, mito-chondria; r, endoplasmic reticulum.

G. A. EDWARDS and H. RUSKA

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Edwards and Ruska—Function and Structure of Insect Muscles 155

and mitochondria form a continuous system in which lie the discontinuousfibrils.

In the sections of the white muscles (figs. 5-8) few tracheoles were observed.They penetrated the sarcolemma, thus entering the fibres, at relatively few

FIG. 8. White fibre of Dytiscus. One tracheole beneath the sarcolemma. Endoplasmicreticulum between the myofibrils, accumulating in the I region, where small mitochondria

can be seen.

legions along its length. In the white muscle of Hydrophilus relatively fewcross-sections of tracheoles were seen among the mitochondria, which some-times occurred in rows between the myofibrils. In the white muscle ofDytiscus only subsarcolemmal tracheoles were observed.

FIG. 7 (plate). White fibre of Dytiscus. Electron micrograph. Fibrils; m, mitochondria,close to the Z bands; r, endoplasmic reticulum.

156 Edwards and Ruska—Function and Structure of Insect Muscles

In no muscle were tracheoles seen to enter the myofibrils or to have adefinite relation to the muscle bands.

Loci of oxidations

The outstanding characteristic of the red flight-muscle of these two insectsis the number and size of the sarcosomes, i.e. the mitochondria of insectmuscles.

In the Hydrophilus flight-muscle the mitochondria are found in rowsparallel to and between the myofibrils and closely associated with the numeroustracheole branches throughout the entire fibre. Just beneath the sarcolemmathere are few mitochondria, relatively speaking, scattered among the largertracheolar branches. Within the body of the fibre, however, internal to theperipheral fibrils, the mitochondria are extremely numerous, one usuallytouching upon the next, separated from the fibrils in most cases by somedistance. In number they average about one mitochondrion to each fibrilsegment. The Hydrophilus mitochondria are largely ovoid or spherical,averaging 2-2 X 1*5/x. The internal structure of the mitochondria appearslike a pile of irregular corrugated cardboard. When cut normal to the planesone sees straight to wavy lines, usually oriented at right angles to the alignmentof the myofilaments. This pattern is interrupted by lacunae, sometimesopening to the surface. When cut with the planes, the mitochondria moreoften present the lacunar picture. It may be possible that the tracheoles incontact with the mitochondria open into the lacunar system.

In the red flight-muscle fibre of Dytiscus the mitochondria appear to bemore numerous, much more closely packed, of the same size generally(averaging 2-5X1-5/A), and often compressed into polygonal shapes withclearly marked limiting membranes. As in Hydrophilus a row of mitochondriais found between the sarcolemma and the peripheral fibrils, and thereafterthere are single or double rows between and parallel to the succeedingmyofibrils. In number they appear to be about 2-3 per segment, tightlysqueezed one against the other. Inasmuch as the tracheoles in this muscleare larger in diameter, fewer in number, and longitudinally arranged betweenthe fibrils, the picture of a mitochondria-tracheole continuous system is lessclearly seen here than in the Hydrophilus muscle fibre. However, the tracheolesagain appear to be more correlated with the mitochondria than with themyofibrils. The internal structure of the mitochondria of the red muscle ofDytiscus, although fundamentally similar to that of Hydrophilus, is muchfiner and is visible only in exceptionally thin sections with good resolution.

Characteristic of the Dytiscus flight muscle, and giving the sections achecker-board appearance, are numerous spherical bodies of uniform size,slightly smaller than the mitochondria described above and scattered amongthem. The bodies seem to be more often associated with the I than the Aregion of the fibril, and occur on the average as one such body to each fibrilsegment. A definite limiting membrane is visible between any two of thesebodies. Internally the structure is similar to that of the other mitochondria,

Edwards and Ruska—Function and Structure of Insect Muscles 157

but shows a looser and thinner arrangement. These bodies can be interpretedas either mitochondria which have undergone a physiological change, or elsea different type of mitochondria. We are more inclined to the former view,believing that this could represent a reversible metabolic state or irreversibleageing.

The white coxal muscles characteristically have very few mitochondria andthese are of small size. The outstanding characteristic of the white musclesis the endoplasmic reticulum. The Hydrophilus white muscle shows greatsimilarity to the mammalian muscle structure shown by Ruska (1954). Themitochondria are aligned in longitudinal rows separating bundles of myofibrils,the fibrils themselves being separated by the endoplasmic reticulum. InDytiscus the mitochondria are arranged singly in transverse rows, i.e. alwaysat the level of the I band between the individual fibrils. In the white musclesof both insects the internal structure of the mitochondria appears to besimilar to but less distinct than that of the mitochondria of the red musclefibres. In form they are roughly oval, averaging 0-4 X I-O/A.

Endoplasmic reticulumThe endoplasmic reticulum is peculiar to the white muscles but apparently

lacking or much less developed in the flight-muscle fibres. It appears to bea continuous system located between the myofibrils and connected to themat the level of the I region. Thus in low magnifications a longitudinal sectionthrough a white fibre shows an apparently continuous Z line. On amplifica-tion, however, one can see that the Z is actually restricted to the myofibrilbut that connexion of one fibril to the next is made by the reticular system.In Hydrophilus the system appears to be represented by dark threads betweenthe fibrils, sending very fine branches between the myofilaments predomin-antly at the level of the I region. In Dytiscus, on the other hand, the systemis definitely tubular and appears rather to surround the fibrils than to enterthem. The greatest concentration of tubes is at the Z line, but a secondaryaccumulation occurs near the middle of each segment. In the red muscleof Hydrophilus faint traces of endoplasmic material may be seen in thevicinity of Z.

Myofibrils

The sections of the fibrils confirm the older findings concerning thedifferences between red and white insect muscles. The red fibrils in Hydro-philus are more uniform in diameter and are more clearly separated than thewhite fibrils. Actually in the white muscle fibres the fibrils form a synfibrillarcontinuous system. In all fibrils of both insects the myofilaments are clearlyvisible; they are continuous, at least in the contracted state, throughoutboth A and I regions. The filaments of the white muscle of Hydrophilusappear to be more loosely packed and in less orderly arrangement than inthe red and the white muscles of Dytiscus. In both types of muscle it couldbe seen that the fibrils are certainly composed of more than just filaments.

158 Edwards and Ruska—Function and Structure of Insect Muscles

Further details of their structure will be discussed elsewhere. It should benoted here, however, that only in the Hydrophilus white muscle was the Zline seen to be attached to the sarcolemma, giving it a scalloped form.

DISCUSSION

The results have shown very clearly that the specialized functions of themuscles studied are based upon specialization in structure. The flight-muscle,with its high velocity of movement and its high oxygen consumption, possessesan intracellular tracheole system closely linked to large numbers of hugemitochondria, thus providing the carriers of the oxidative enzymes with anample oxygen supply. The flight-muscle is therefore essentially a fast-actingenergy-producing machine. The oxidative mechanism is very close to thecontractile mechanism. The loci of oxidations completely surround eachfibril. This is a logical mechanism inasmuch as the flight-muscle fibril musthave energy available to it, must have end products removed, and must getback resynthesized carbohydrates and phosphates as rapidly as possible forrepetitive contractions. To this end strength has been sacrificed, most of thefibre space being occupied by the mitochondria-tracheole system.

The white muscle is slower, capable of continuous tension, has a loweroxygen consumption, but needs a continuous low energy supply. The greaterpart of the fibrillar areas is filled with the contractile substance, the mito-chondria occupying a very small fraction of the space. Actually this musclehas less mitochondria and tracheolization than would be expected on thebasis of its metabolic activity, but we musf keep in mind that the centralpart of the white Dytiscus muscle contains more cytoplasmic material. Theremaining space between the fibrils is occupied by the endoplasmic reticulum.The white muscles apparently are constructed for strength rather than fastaction and have therefore sacrificed oxidative capacity.

The presence of the endoplasmic reticulum in the white muscle and themitochondria-tracheole system in the red muscle raises the question of theirrespective roles in the metabolism and functions of these muscles. Themitochondrial system is predominant in mammalian diaphragm (Ruska, 1954),in vertebrate heart-muscle (Kisch and Philpott, 1953), and insect flight-muscle. The endoplasmic reticulum has been found to occur in the breastmuscle of the hen (Bennett and Porter, 1953), mammalian leg muscles(Ruska, 1954), and in insect white muscles. Thus the mitochondrial systemappears to be linked to the need for repeated bursts of energy for fast andstrong action, whereas the basiphil reticular system is more suitable for themaintenance of tension where more time is available for resynthesis.

We are very grateful to Dr. Aristides Vallejo-Freire, of the Seccao de Virusof the Instituto Butantan, who placed at our disposal so many of the facilitiesnecessary for the work. We wish to thank also Dr. Keith R. Porter and

Edwards and Ruska—Function and Structure of Insect Muscles 159

Mr. G. Gilcher, of the Rockefeller Institute for Medical Research, who madeavailable the microtome used in this study. The work was supported in partby a grant from the Conselho Nacional de Pesquisas, Rio de Janeiro, Brazil.

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Ent. Soc. Amer., 47, 343.19546. Ibid., 47, 459.

HOLMGREN, E., 1909. Scand. Arch. Physiol., s i , 287.1913. Anatom. Anzeiger, 44, 225.

KISCH, B., and PHILPOTT, D. E., 1953. Exp. Med. and Surgery, 11, 161.KNOLL, P., 1889. Sitzungsber. Akad. d. Wissensch. Wien, 98, 456.PALADE, G. E., 1952. J. exp. Med., 95, 285.PEREZ GONZALEZ, M. D., and EDWARDS, G. A., 1954. Bol. Fac. Fil. Cien. Letr., Univ. Sao

Paulo, Zoologia, 19, 371.ROEDER,' K. D., 1951. Biol. Bull., 100, 95.RUSKA, H., 1954. Zeit. f. Naturforsch., 96, 358.